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Melatonin reduces formalin-induced nociception and tactile allodynia in diabetic rats
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European Journal of Pharmacology 577 (2007) 203 – 210 www.elsevier.com/locate/ejphar
Melatonin reduces formalin-induced nociception and tactile allodynia in diabetic rats Rosaura Arreola-Espino a , Héctor Urquiza-Marín a , Mónica Ambriz-Tututi b , Claudia Ivonne Araiza-Saldaña b , Nadia L. Caram-Salas b , Héctor I. Rocha-González b , Teresa Mixcoatl-Zecuatl c , Vinicio Granados-Soto b,c,⁎ a
Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico b Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico c Centro de Investigación y de Estudios Avanzados, Unidad Monterrey, Monterrey, Nuevo León, Mexico Received 19 March 2007; received in revised form 17 September 2007; accepted 18 September 2007 Available online 24 September 2007
Abstract The purpose of this study was to assess the antinociceptive and antiallodynic effect of melatonin as well as its possible mechanism of action in diabetic rats. Streptozotocin (50 mg/kg) injection caused hyperglycemia within 1 week. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Oral administration of melatonin (10–300 mg/kg) dose-dependently reduced flinching behavior in diabetic rats. In addition, K-185 (a melatonin MT2 receptor antagonist, 0.2–2 mg/kg, s.c.) completely blocked the melatonin-induced antinociception in diabetic rats, whereas that naltrexone (a non-selective opioid receptor antagonist, 1 mg/kg, s.c.) and naltrindole (a selective δ opioid receptor antagonist, 0.5 mg/kg, s.c.), but not 5′-guanidinonaltrindole (a selective κ opioid receptor antagonist, 1 mg/kg, s.c.), partially reduced the antinociceptive effect of melatonin. Given alone K-185, naltrexone, naltrindole or 5′-guanidinonaltrindole did not modify formalin-induced nociception in diabetic rats. Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats. On this condition, oral administration of melatonin (75–300 mg/kg) dose-dependently reduced tactile allodynia in diabetic rats. Both antinociceptive and antiallodynic effects were not related to motor changes as melatonin did not modify number of falls in the rotarod test. Results indicate that melatonin is able to reduce formalin-induced nociception and tactile allodynia in streptozotocin-injected rats. In addition, data suggest that melatonin MT2 and δ opioid receptors may play an important role in these effects. © 2007 Elsevier B.V. All rights reserved. Keywords: Melatonin; Melatonin MT2 receptor; Naltrexone; Naltrindole; Diabetes; Tactile allodynia
1. Introduction Diabetes mellitus is one of the most common chronic medical conditions affecting over 100 million people worldwide, of whom up to 60% may develop diabetic neuropathy (Galer et al., 2000). The treatment of pain in diabetic patients is frequently unsatisfactory. Anticonvulsants, tricyclic antidepressants and opioids have become the mainstay in the treatment of ⁎ Corresponding author. Cinvestav, Unidad Monterrey, Avenida Cerro de las Mitras 2565, Colonia Obispado, 64060 Monterrey, Nuevo León, Mexico. Tel.: +52 81 8220 1740; fax: +52 81 8220 1741. E-mail address: [email protected] (V. Granados-Soto). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.09.006
chronic neuropathic pain (Sindrup and Jensen, 1999). However, these drugs often have a limited effect or may cause intolerable side effects. Therefore, other options of treatment are needed. Melatonin (N-acethyl-5-methoxytryptamine) is a hormone synthesized primarily in the mammalian pineal gland and secreted into the bloodstream (Vanecek, 1998). This hormone is involved in several biological functions, including circadian rhythms, sleep and analgesia (Morgan et al., 1994; Vanecek, 1998; von Gall et al., 2002; Simonneaux and Ribelayga, 2003; Zahn et al., 2003). Melatonin has been extensively studied in inflammatory pain models in animals (Cuzzocrea et al., 1997; Raghavendra et al., 2000; Pang et al., 2001; Bilici et al., 2002; El-Shenawy et al., 2002; Ray et al., 2004; Wang et al., 2006).
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Moreover, there is also evidence about its efficacy in neuropathic pain models (Ulugol et al., 2006; Ambriz-Tututi and Granados-Soto, in press). Melatonin has shown to reduce several diabetes-induced complications in animals such as oxidative–antioxidative status (Anwar and Meki, 2003; Aksoy, 2003) and the pancreatic (Kanter et al., 2006), renal (Derlacz et al., 2007) and liver (Guven et al., 2006) injury. However, its efficacy in hyperalgesia and tactile allodynia in diabetic rats has not been assessed. It is believed that diabetes-induced hyperglycemia causes neural degeneration via the increased oxidative stress (Montilla et al., 1998; Nishida, 2005). Thus, we have hypothesized that melatonin could reduce formalin-induced nociception and tactile allodynia in diabetic animals. Therefore, the purpose of this work was to study the possible antinociceptive and antiallodynic effect of melatonin as well as its mechanism of action in diabetic rats. 2. Materials and methods 2.1. Animals Experiments were performed on adult female Wistar rats (body weight range, 220–240 g) of 10–12 weeks of age. The animals were obtained from our own breeding facilities and had free access to drinking water, but food was withdrawn 12 h before experiments. Under this condition, we observed that streptozotocin produced a greater % of diabetic rats (90%). Experiments were done in normal light/dark cycle and they were started at the same time (10:00 AM). Efforts were made to minimize animal suffering and to reduce the number of animals used. All experiments followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann, 1983) and were approved by our local Ethics Committee. 2.2. Induction of diabetes Rats were injected with streptozotocin (50 mg/kg, i.p.) (Sigma, St. Louis, MO, USA) to produce experimental diabetes (Courteix et al., 1993). Control animals (age-matched) received distilled water. Diabetes was confirmed 1 week after injection by measurement of tail vein blood glucose levels with the glucose meter Ascensia ELITE (Bayer, Mexico City). Two weeks after streptozotocin injection, glycemia was again determined and only animals with a final blood glucose level ≥ 300 mg/dl were included in the study (90%). 2.3. Assessment of nociception Nociception in non-diabetic and diabetic (2 weeks) rats was assessed using the 0.5% formalin test (Araiza-Saldaña et al., 2005; Juárez-Rojop et al., 2006). The rats were placed in open plexiglas observation chambers for 30 min to allow them to acclimate to their surroundings; then they were removed for formalin administration. Fifty μl of diluted formalin (0.5%) was injected subcutaneously into the dorsal surface of the right hind paw with a 30-gauge needle. The animals were returned to the chambers and nociceptive behavior was observed immediately after formalin
injection. Mirrors were placed in each chamber to enable unhindered observation. Nociceptive behavior was quantified as the number of flinches of the injected paw during 1-min periods every 5 min, up to 60 min after injection (Wheeler-Aceto and Cowan, 1991). Flinching was readily discriminated and was characterized as a rapid and brief withdrawal, or as a flexing of the injected paw. Formalin-induced flinching behavior was biphasic (Dubuisson and Dennis, 1977; Araiza-Saldaña et al., 2005). The initial acute phase (0–10 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–60 min). Animals were used only once and at the end of the experiment they were sacrificed in a CO2 chamber. 2.4. Assessment of allodynia Tactile allodynia was tested in diabetic rats 4 to 8 weeks after streptozotocin injection as previously described (Chaplan et al., 1994; Sánchez-Ramírez et al., 2006). Briefly, rats were transferred to a clear plastic, wire mesh-bottomed cage and allowed to acclimatize for 30 min. von Frey filaments (Stoelting, Wood Dale, IL) were used to determine the 50% paw withdrawal threshold using the up–down method of Dixon (1980). A series of filaments, starting with one that had a buckling weight of 2 g, was applied in consecutive sequence to the plantar surface of the right hind paw with a pressure causing the filament to buckle. Lifting of the paw indicated a positive response and prompted the use of the next weaker filament whereas that absence of a paw withdrawal after 5 s indicated a negative response and prompted the use of the next filament of increasing weight. This paradigm continued until four more measurements had been made after the initial change of the behavioral response or until 5 consecutive negative (assigned a score of 15 g) or four consecutive positive (assigned a score of 0.25 g) responses had occurred. The resulting scores were used to calculate the 50% response threshold by using the formula: 50% g threshold = 10(Xf + κ∂) / 10,000, where Xf = the value (in log units) of the final von Frey filament used, κ = the value (from table in Chaplan et al., 1994) for the pattern of positive and/or negative responses, and ∂ = the mean difference (in log units) between stimulus strengths. Behavioral tests were performed immediately before and 60 min after drug administration. Threshold was then assessed every 30 min until 4 h. Allodynia was considered to be present when paw withdrawal thresholds were b 4 g. Diabetic rats not demonstrating allodynia were not further studied. 2.5. Drugs Streptozotocin, melatonin (N-acethyl-5-methoxytryptamine), K-185 (N-butanoyl 2-(5,6,7-trihydro-11-methoxybenzo[3,4] cyclohept[2,1-a]indol-13-yl)ethanamine), naltrexone, naltrindole and 5′-guanidinonaltrindole were obtained from Sigma (St. Louis, MO, USA). Streptozotocin was freshly dissolved in distilled water (obtained from a Milli-Q water system), protected from light and immediately administered. Melatonin was dissolved in carboxymethyl cellulose 1% and given orally by a gastric tube at a volume ratio of 4 ml/kg. K-185 was dissolved in 20% dimethylsulfoxide (DMSO) while that naltrexone, naltrindole and 5′-guanidinonaltrindole were dissolved in 0.9% isotonic saline.
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2.6. Study design Independent groups of animals (n = 6) were used for each experimental condition. In order to define the best time to administer melatonin, diabetic rats received an oral administration of vehicle (carboxymethyl cellulose 1%) or melatonin (150 mg/ kg, p.o.) at 20 and 60 min before formalin injection (50 μl). These times were selected from the literature (El-Shenawy et al., 2002) and from pilot experiments in our laboratory. Since the best antinociceptive effect was observed at the 60-min pretreatment (data not shown), dose–response curve for melatonin was carried out giving vehicle or increasing doses of melatonin (10–300 mg/ kg, p.o.) 60 min before formalin injection into the right paw. In an attempt to determine the possible mechanism of antinociceptive action of melatonin in diabetic rats, the selective melatonin MT2 receptor antagonist K-185 (0.2–2 mg/kg, s.c.) was administered 15 min before melatonin (150 mg/kg, p.o.) administration. To establish whether melatonin-induced antinociception was mediated by opioid receptors activation, the effect of the non-selective opioid receptor antagonist naltrexone (– 70 min, 1 mg/kg, s.c.) on the antinociceptive activity induced by melatonin (− 60 min, 150 mg/kg, p.o.) was assessed. Furthermore, to define the nature of the opioid receptor involved in the antinociceptive effect of melatonin in diabetic rats, the selective δ (naltrindole, 0.5 mg/kg, s.c.) and κ (5′guanidinonaltrindole, 1 mg/kg, s.c.) opioid receptor antagonists were given 10 min before melatonin (150 mg/kg, p.o.). Melatonin MT2 receptor antagonist K-185 (Faust et al., 2000) and the δ and κ opioid receptor antagonists naltrindole (Portoghese et al., 1988) and 5′-guanidinonaltrindole (Jones and Portoghese, 2000), respectively, were chosen by their high affinity for the studied receptors. In addition, doses of these drugs were selected from previous studies (Reeta et al., 2006; Qi et al., 2006) and from pilot experiments in our laboratory. For the study of allodynia, rats received the oral administration of vehicle (carboxymethyl cellulose 1%) or increasing doses of melatonin (75–300 mg/kg, p.o.) and withdrawal threshold in diabetic rats (4–8 weeks) was measured for the next 4 h. Observer was unaware of the treatment in each animal.
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according to trapezoidal rule. For allodynia, curves were constructed plotting the 50% threshold for paw withdrawal as a function of time. Effect was also expressed as the area under the 50% threshold withdrawal against time curve (AUC). One-way analysis of variance (ANOVA) followed by Tukey's test was used to compare differences between treatments. The Mann–Whitney test was used to assess differences in rats submitted to the rotarod test. Differences were considered to reach statistical significance when P b 0.05. 3. Results 3.1. Streptozotocin injection and formalin-evoked flinching behavior in non-diabetic and diabetic rats Streptozotocin, but not distilled water, injection caused hyperglycemia. The blood glucose level measured in these rats was 61.8 ± 3.7 and 65± 1.9 mg/dl before distilled water, or streptozotocin injection and 89.2 ±0.86 and 368.6± 14.8 mg/dl 15 days after saline or streptozotocin injection (5 rats), respectively. Moreover, these rats had an increase in food and water intake and displayed polyuria (data not shown). Both the streptozotocin-induced diabetic (2 weeks) and nondiabetic groups exposed to 0.5% formalin exhibited the biphasic pattern of this test characterized by periods of flinching of the injected hind paw separated by a period of decreased activity. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats (Fig. 1A). The overall analysis of formalin-evoked flinching as AUC, showed a small but significant difference (P b 0.05) between the diabetic and non-
2.7. Motor co-ordination test Two independent groups of rats (n = 6, each) were examined for motor co-ordination in a treadmill apparatus (rotarod test) before and after receiving melatonin 300 mg/kg or vehicle by oral administration. Animals were placed upon a cylinder (7 cm diameter) rotating at a speed of 10 rpm. Rats were trained to walk on the cylinder for three consecutive sessions and on the fourth, they received the drug or vehicle treatment (time 0) and the number of falls during a 5-min period was counted after 45 min. 2.8. Data analysis and statistics All results are presented as the mean ± S.E.M. for 6 animals per group. For the formalin test, curves were made for the mean number of flinches against time. The area under the number of flinches against time curves (AUC) for both phases was calculated
Fig. 1. A) Time course of the nociceptive behavior induced by subcutaneous injection of 0.5% formalin to non-diabetic (white circles) and diabetic (black circles) rats. Panels B and C show formalin-induced nociceptive behavior, in non-diabetic and diabetic rats during phases 1 and 2, respectively. The later data are expressed as the area under the number of flinches against time curve (AUC). Data are the mean ± S.E.M. of 6 animals. ⁎Significantly different from the nondiabetic group (P b 0.05), as determined by Student′s t-test.
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diabetic groups during the first phase (Fig. 1B). Moreover, additional analysis revealed that the diabetic group had significantly (P b 0.05) increased frequency of flinching during the second phase (Fig. 1C). Since streptozotocin mainly increased nociception during phase 2 of the formalin test, in the following studies only phase 2 was further analyzed. 3.2. Antinociceptive effect of melatonin in diabetic rats Oral administration of melatonin (150 mg/kg) significantly (P b 0.05) reduced formalin-induced nociceptive behavior at 20and 60-min pretreatment. However, the best antinociceptive effect was observed with the 60-min pretreatment (data not shown) and this pretreatment time was used in the following experiments. Under this condition, melatonin significantly (Fig. 2A, P b 0.05) and dose-dependently (Fig. 2B) reduced flinching behavior during phase 2 in diabetic rats. 3.3. Effect of K-185 and naltrexone on the antinociceptive effect of melatonin in diabetic rats Subcutaneous administration of K-185 (0.2–2 mg/kg) blocked in a dose-related manner the melatonin-induced antinociceptive activity in diabetic rats (Fig. 3A). Moreover, naltrexone (1 mg/kg, s.c.) partially reduced the antinociceptive effect of melatonin in diabetic rats (2 weeks, Fig. 3B). At the greatest tested doses, K-185 or naltrexone did not modify the formalin-induced nociception in diabetic rats (Fig. 3).
Fig. 3. Effect of the melatonin MT2 receptor antagonist K-185 (A) and the nonselective opioid receptor antagonist naltrexone (NTX, B) on the antinociceptive effect of melatonin in diabetic rats submitted to the 0.5% formalin test. Data are expressed as the area under the curve of the number of flinches against time (AUC) of phase 2. Bars are the mean ± S.E.M. for 6 animals. ⁎Significantly different from the vehicle group (P b 0.05) and #significantly different from melatonin group, as determined by analysis of variance followed by Tukey's test.
3.4. Effect of naltrindole and 5′-guanidinonaltrindole on the antinociceptive effect of melatonin in diabetic rats Subcutaneous administration of the selective δ (naltrindole) and κ (5′-guanidinonaltrindole) opioid receptor antagonists did not modify formalin-induced flinching behavior in diabetic rats (Fig. 4). However, naltrindole partially but significantly (P b 0.05) diminished the antinociceptive effect of melatonin in diabetic rats (Fig. 4A). In marked contrast, the selective κ opioid receptor antagonist 5′-guanidinonaltrindole was not able to modify the antinociceptive effect of melatonin in diabetic rats (Fig. 4B). 3.5. Antiallodynic effect of melatonin in diabetic rats
Fig. 2. A) Time course of the antinociceptive effect of melatonin (300 mg/kg, p. o.) in diabetic rats submitted to the subcutaneous injection of 0.5% formalin. Panel B shows the dose–response curve for the antinociceptive effect of melatonin in diabetic rats. In this plot, data are expressed as the area under the curve of the number of flinches against time (AUC) of phase 2. Bars are the mean ± S.E.M. for 6 animals. ⁎Significantly different from the vehicle group (P b 0.05), as determined by analysis of variance followed by Tukey's test.
Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats compared to the distilled water-injected rats. No autotomy behavior was ever observed during the experiment. On this condition, oral administration of melatonin (300 mg/kg), but not vehicle (carboxymethyl cellulose 1%), increased the withdrawal threshold in diabetic rats (Fig. 5A). The maximal antiallodynic effect was reached at 1.5 h and it lasted for 4 h with the greatest dose tested (300 mg/kg). In addition, melatonin dose-dependently reduced streptozotocin-induced tactile allodynia (P b 0.05) (Fig. 5B).
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Moreover, melatonin is able to reduce tactile allodynia induced by ligation of L5/L6 spinal nerves (Ambriz-Tututi and GranadosSoto, in press) and by thermal hyperalgesia in neuropathic mice with partial tight ligation of the sciatic nerve (Ulugol et al., 2006). Thus, our data confirm these studies and extend these observations by showing that melatonin is able to reverse formalininduced flinching behavior, tactile allodynia and abnormal pain processing mechanisms present in diabetic rats. To our knowledge, this is the first report about the antinociceptive and antiallodynic effect of melatonin in streptozotocin-injected diabetic rats. 4.2. Effect of K-185, naltrexone, naltrindole or 5′guanidinonaltrindole on the antinociceptive effect of melatonin in diabetic rats
Fig. 4. Effect of the selective δ (A) and κ (B) opioid receptor antagonists naltrindole and 5′-guanidinonaltrindole (GNTI), respectively, on the antinociceptive effect of melatonin in diabetic rats submitted to the 0.5% formalin test. Data are expressed as the area under the curve of the number of flinches against time (AUC) of phase 2. Bars are the mean ± S.E.M. for 6 animals. ⁎Significantly different from the vehicle group (P b 0.05) and #significantly different from melatonin group, as determined by analysis of variance followed by Tukey's test.
The mechanisms of antinociceptive and antiallodynic activity of melatonin in diabetic rats are unknown. In an attempt to find the possible mechanisms of antinociceptive action, the effect of K-185 and naltrexone on the antinociceptive effect of melatonin in diabetic rats submitted to the 0.5% formalin test was assessed. The antinociceptive effect of melatonin was diminished by K-185. Since K-185 is a highly selective melatonin MT2 receptor antagonist (Faust et al., 2000),
3.6. Effect of melatonin on motor co-ordination test Animals did not have a fall from the rotarod test before administration of melatonin or saline. Oral administration of melatonin (300 mg/kg) produced 1 fall in 1 of 6 rats during the rotarod test. Moreover, oral administration of vehicle showed the same pattern (data not shown). 4. Discussion 4.1. Antinociceptive and antiallodynic activity of melatonin in diabetic rats In this study we have shown that oral administration of melatonin is able to reduce formalin-induced nociceptive behavior and tactile allodynia in diabetic rats. This antinociceptive and antiallodynic effect was independent of any motor effect as administration of the greatest systemic tested dose (300 mg/kg, p.o.) significantly reduced formalin-induced nociception and tactile allodynia without affecting co-ordination in the rotarod test, as previously reported (Tu et al., 2004; Ambriz-Tututi and Granados-Soto, in press). Several studies have shown that melatonin produces antinociception in inflammatory pain models (Cuzzocrea et al., 1997; Raghavendra et al., 2000; Bilici et al., 2002; El-Shenawy et al., 2002; Ray et al., 2004; Tu et al., 2004).
Fig. 5. A) Time course of the antiallodynic effect of melatonin (300 mg/kg, p.o.) in streptozotocin-pretreated diabetic rats. After diabetes induction, animals were allowed to develop tactile allodynia for 4 to 8 weeks. Animals were treated with oral melatonin and withdrawal threshold was measured for the next 4 h. B) Antiallodynic effect produced by oral administration of melatonin in diabetic (4–8 weeks) rats. Data are expressed as the 50% threshold withdrawal against time curve (AUC). ⁎Significantly different from the vehicle (Veh) group (P b 0.05), as determined by analysis of variance followed by the Tukey's test. In both plots data are the mean ± S.E.M. for 6 animals.
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our data suggest that melatonin-induced antinociception could be due to activation of melatonin MT2 receptors. These results agree with the presence of melatonin MT2 receptors in the dorsal horn of the lumbar spinal cord (Zahn et al., 2003). In addition, our data agree with an inhibitory role for melatonin MT2 receptors as there is evidence that binding of melatonin to melatonin MT2 receptors decreases intracellular concentrations of cyclic AMP, diacylglycerol and arachidonic acid (Iuvone and Gan, 1994; Vanecek, 1998). On the other hand, subcutaneous administration of the nonselective opioid receptor antagonist naltrexone partially diminished the antinociceptive effect induced by the oral administration of melatonin. These data agree with previous observations showing that melatonin-induced antinociceptive and antihyperalgesic (Mantovani et al., 2006) or antiallodynic (Ambriz-Tututi and Granados-Soto, in press) effects in nondiabetic rats can be blocked by the non-selective opioid receptor antagonist naloxone or naltrexone, respectively. In an attempt to define the nature of the opioid receptors activated after oral administration of melatonin, the selective δ and κ opioid receptor antagonists naltrindole (Portoghese et al., 1988) and 5′guanidinonaltrindole (Jones and Portoghese, 2000), respectively, were used alone or in combination with melatonin in diabetic rats. Likewise naltrexone, naltrindole, but not 5′-guanidinonaltrindole, partially reduced melatonin-induced antinociception. Accordingly, it has been demonstrated that intraperitoneal or intracerebroventricular administration of melatonin significantly enhances the δ opioid receptor agonist deltorphin I-induced antinociception, but not that induced by the μ opioid receptor agonist endomorphin-1 (Li et al., 2005). This effect was blocked by the melatonin MT2 receptor antagonist luzindol suggesting that melatonin can augment antinociception induced by the δ opioid receptor agonists acting on melatonin MT2 receptors. Taken together data suggest the possible participation of δ, but not κ, opioid receptors in the antinociceptive effect of melatonin in diabetic rats. It is well known that melatonin is not able to bind opioid receptors (Shavali et al., 2005). However, several observations, including our data, point to significant interactions between melatonin and opioid peptides (Shavali et al., 2005). Melatonin releases β-endorphin, an endogenous opioid peptide, from mouse pituitary cells in culture (Shavali et al., 2005). Hence, it has been suggested that melatonin exerts its antinociceptive action in diabetic rats not by binding to opioid receptors but by inducing β-endorphin release. β-endorphin could then bind to δ (Aloyo and Pazdalski, 1995) or μ (Gilmore and Weiner, 1989) opioid receptors in spinal cord and supraspinally (periaqueductal gray matter) in order to produce its antinociceptive effect. This suggestion agrees with evidence showing that β-endorphin administered i.c.v. produces a naltrindolesensitive antinociceptive effect in diabetic mice (Kamei et al., 1993). In addition, the antinociceptive effect of melatonin in spinal and supraspinal sites in non-diabetic rats also supports this suggestion (Yu et al., 2000a,b; Tu et al., 2004; AmbrizTututi and Granados-Soto, in press). All these sites would be reached considering the high lipid-solubility of melatonin. The final effect would be the opening of K+ channels and closing of
Ca2+ channels (Moises et al., 1994; Kieffer, 1995; Lohmann and Welch, 1999) leading to cell hyperpolarization and reduction of pain. In this study we have provided evidence about the possible mechanisms involved in the antinociceptive actions of melatonin in diabetic rats submitted to the 0.5% formalin test. It is likely that some of these mechanisms could work to explain the antiallodynic activity of this drug. However, the experiments demonstrating these mechanisms are still lacking. 4.3. Final considerations Diabetes-induced hyperglycemia plays a critical role in the development and progression of diabetic neuropathy. One of the mechanisms by which hyperglycemia causes neural degeneration is via the increased oxidative stress that accompanies diabetes (Montilla et al., 1998; Baydas et al., 2003; Nishida, 2005). Hyperglycemia causes a reduction in the levels of protective endogenous antioxidants and increases generation of free radicals (Baydas et al., 2002). Contrariwise, melatonin is a direct scavenger of free radicals and has indirect antioxidative effects due to the stimulation of the expression and activity of antioxidative enzymes such as glutathione peroxidase, superoxide dismutase and catalase in mammalian cells (Nishida, 2005; Klepac et al., 2006). Therefore, it is tempting to suggest that these actions could explain at least part of the antinociceptive and/or antiallodynic effect in diabetic rats. The fact that systemic administration of free radical scavengers is able to reduce tactile allodynia in the spinal nerve ligation model in rats (Kim et al., 2004) agrees with our suggestion. In conclusion, oral administration of melatonin to diabetic rats significantly reduces formalin-induced nociception and tactile allodynia. The antinociceptive effects are completely prevented by the melatonin MT2 receptor antagonist K-185 and partially prevented by the non-selective opioid receptor antagonist naltrexone and the selective δ opioid receptor antagonist naltrindole, but not by the selective κ opioid receptor antagonist 5′-guanidinonaltrindole. Thus, data suggest the possible participation of melatonin MT2 and δ opioid receptors in the antinociceptive activity of melatonin in diabetic rats. Some of these mechanisms, along with the ability to scavenge free radicals, could also explain the antiallodynic effect shown by this drug. Finally, our data suggest that melatonin could be a promising drug to treat diabetic patients. Acknowledgements This work was done at the Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico. Authors greatly appreciate the technical assistance of Guadalupe C. Vidal-Cantú and bibliographic assistance of Héctor Vázquez. Rosaura Arreola-Espino, Mónica Ambriz-Tututi, Claudia I. Araiza-Saldaña, Nadia L. CaramSalas and Héctor I. Rocha-González are CONACYT fellows. In addition, Claudia I. Araiza-Saldaña and Nadia L. Caram-Salas are the recipients of the “Apoyos Integrales para la Formación de Doctores en Ciencias” fellowship. Teresa Mixcoatl-Zecuatl is a
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European Journal of Pharmacology 577 (2007) 203 – 210 www.elsevier.com/locate/ejphar
Melatonin reduces formalin-induced nociception and tactile allodynia in diabetic rats Rosaura Arreola-Espino a , Héctor Urquiza-Marín a , Mónica Ambriz-Tututi b , Claudia Ivonne Araiza-Saldaña b , Nadia L. Caram-Salas b , Héctor I. Rocha-González b , Teresa Mixcoatl-Zecuatl c , Vinicio Granados-Soto b,c,⁎ a
Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico b Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico c Centro de Investigación y de Estudios Avanzados, Unidad Monterrey, Monterrey, Nuevo León, Mexico Received 19 March 2007; received in revised form 17 September 2007; accepted 18 September 2007 Available online 24 September 2007
Abstract The purpose of this study was to assess the antinociceptive and antiallodynic effect of melatonin as well as its possible mechanism of action in diabetic rats. Streptozotocin (50 mg/kg) injection caused hyperglycemia within 1 week. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Oral administration of melatonin (10–300 mg/kg) dose-dependently reduced flinching behavior in diabetic rats. In addition, K-185 (a melatonin MT2 receptor antagonist, 0.2–2 mg/kg, s.c.) completely blocked the melatonin-induced antinociception in diabetic rats, whereas that naltrexone (a non-selective opioid receptor antagonist, 1 mg/kg, s.c.) and naltrindole (a selective δ opioid receptor antagonist, 0.5 mg/kg, s.c.), but not 5′-guanidinonaltrindole (a selective κ opioid receptor antagonist, 1 mg/kg, s.c.), partially reduced the antinociceptive effect of melatonin. Given alone K-185, naltrexone, naltrindole or 5′-guanidinonaltrindole did not modify formalin-induced nociception in diabetic rats. Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats. On this condition, oral administration of melatonin (75–300 mg/kg) dose-dependently reduced tactile allodynia in diabetic rats. Both antinociceptive and antiallodynic effects were not related to motor changes as melatonin did not modify number of falls in the rotarod test. Results indicate that melatonin is able to reduce formalin-induced nociception and tactile allodynia in streptozotocin-injected rats. In addition, data suggest that melatonin MT2 and δ opioid receptors may play an important role in these effects. © 2007 Elsevier B.V. All rights reserved. Keywords: Melatonin; Melatonin MT2 receptor; Naltrexone; Naltrindole; Diabetes; Tactile allodynia
1. Introduction Diabetes mellitus is one of the most common chronic medical conditions affecting over 100 million people worldwide, of whom up to 60% may develop diabetic neuropathy (Galer et al., 2000). The treatment of pain in diabetic patients is frequently unsatisfactory. Anticonvulsants, tricyclic antidepressants and opioids have become the mainstay in the treatment of ⁎ Corresponding author. Cinvestav, Unidad Monterrey, Avenida Cerro de las Mitras 2565, Colonia Obispado, 64060 Monterrey, Nuevo León, Mexico. Tel.: +52 81 8220 1740; fax: +52 81 8220 1741. E-mail address: [email protected] (V. Granados-Soto). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.09.006
chronic neuropathic pain (Sindrup and Jensen, 1999). However, these drugs often have a limited effect or may cause intolerable side effects. Therefore, other options of treatment are needed. Melatonin (N-acethyl-5-methoxytryptamine) is a hormone synthesized primarily in the mammalian pineal gland and secreted into the bloodstream (Vanecek, 1998). This hormone is involved in several biological functions, including circadian rhythms, sleep and analgesia (Morgan et al., 1994; Vanecek, 1998; von Gall et al., 2002; Simonneaux and Ribelayga, 2003; Zahn et al., 2003). Melatonin has been extensively studied in inflammatory pain models in animals (Cuzzocrea et al., 1997; Raghavendra et al., 2000; Pang et al., 2001; Bilici et al., 2002; El-Shenawy et al., 2002; Ray et al., 2004; Wang et al., 2006).
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Moreover, there is also evidence about its efficacy in neuropathic pain models (Ulugol et al., 2006; Ambriz-Tututi and Granados-Soto, in press). Melatonin has shown to reduce several diabetes-induced complications in animals such as oxidative–antioxidative status (Anwar and Meki, 2003; Aksoy, 2003) and the pancreatic (Kanter et al., 2006), renal (Derlacz et al., 2007) and liver (Guven et al., 2006) injury. However, its efficacy in hyperalgesia and tactile allodynia in diabetic rats has not been assessed. It is believed that diabetes-induced hyperglycemia causes neural degeneration via the increased oxidative stress (Montilla et al., 1998; Nishida, 2005). Thus, we have hypothesized that melatonin could reduce formalin-induced nociception and tactile allodynia in diabetic animals. Therefore, the purpose of this work was to study the possible antinociceptive and antiallodynic effect of melatonin as well as its mechanism of action in diabetic rats. 2. Materials and methods 2.1. Animals Experiments were performed on adult female Wistar rats (body weight range, 220–240 g) of 10–12 weeks of age. The animals were obtained from our own breeding facilities and had free access to drinking water, but food was withdrawn 12 h before experiments. Under this condition, we observed that streptozotocin produced a greater % of diabetic rats (90%). Experiments were done in normal light/dark cycle and they were started at the same time (10:00 AM). Efforts were made to minimize animal suffering and to reduce the number of animals used. All experiments followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann, 1983) and were approved by our local Ethics Committee. 2.2. Induction of diabetes Rats were injected with streptozotocin (50 mg/kg, i.p.) (Sigma, St. Louis, MO, USA) to produce experimental diabetes (Courteix et al., 1993). Control animals (age-matched) received distilled water. Diabetes was confirmed 1 week after injection by measurement of tail vein blood glucose levels with the glucose meter Ascensia ELITE (Bayer, Mexico City). Two weeks after streptozotocin injection, glycemia was again determined and only animals with a final blood glucose level ≥ 300 mg/dl were included in the study (90%). 2.3. Assessment of nociception Nociception in non-diabetic and diabetic (2 weeks) rats was assessed using the 0.5% formalin test (Araiza-Saldaña et al., 2005; Juárez-Rojop et al., 2006). The rats were placed in open plexiglas observation chambers for 30 min to allow them to acclimate to their surroundings; then they were removed for formalin administration. Fifty μl of diluted formalin (0.5%) was injected subcutaneously into the dorsal surface of the right hind paw with a 30-gauge needle. The animals were returned to the chambers and nociceptive behavior was observed immediately after formalin
injection. Mirrors were placed in each chamber to enable unhindered observation. Nociceptive behavior was quantified as the number of flinches of the injected paw during 1-min periods every 5 min, up to 60 min after injection (Wheeler-Aceto and Cowan, 1991). Flinching was readily discriminated and was characterized as a rapid and brief withdrawal, or as a flexing of the injected paw. Formalin-induced flinching behavior was biphasic (Dubuisson and Dennis, 1977; Araiza-Saldaña et al., 2005). The initial acute phase (0–10 min) was followed by a relatively short quiescent period, which was then followed by a prolonged tonic response (15–60 min). Animals were used only once and at the end of the experiment they were sacrificed in a CO2 chamber. 2.4. Assessment of allodynia Tactile allodynia was tested in diabetic rats 4 to 8 weeks after streptozotocin injection as previously described (Chaplan et al., 1994; Sánchez-Ramírez et al., 2006). Briefly, rats were transferred to a clear plastic, wire mesh-bottomed cage and allowed to acclimatize for 30 min. von Frey filaments (Stoelting, Wood Dale, IL) were used to determine the 50% paw withdrawal threshold using the up–down method of Dixon (1980). A series of filaments, starting with one that had a buckling weight of 2 g, was applied in consecutive sequence to the plantar surface of the right hind paw with a pressure causing the filament to buckle. Lifting of the paw indicated a positive response and prompted the use of the next weaker filament whereas that absence of a paw withdrawal after 5 s indicated a negative response and prompted the use of the next filament of increasing weight. This paradigm continued until four more measurements had been made after the initial change of the behavioral response or until 5 consecutive negative (assigned a score of 15 g) or four consecutive positive (assigned a score of 0.25 g) responses had occurred. The resulting scores were used to calculate the 50% response threshold by using the formula: 50% g threshold = 10(Xf + κ∂) / 10,000, where Xf = the value (in log units) of the final von Frey filament used, κ = the value (from table in Chaplan et al., 1994) for the pattern of positive and/or negative responses, and ∂ = the mean difference (in log units) between stimulus strengths. Behavioral tests were performed immediately before and 60 min after drug administration. Threshold was then assessed every 30 min until 4 h. Allodynia was considered to be present when paw withdrawal thresholds were b 4 g. Diabetic rats not demonstrating allodynia were not further studied. 2.5. Drugs Streptozotocin, melatonin (N-acethyl-5-methoxytryptamine), K-185 (N-butanoyl 2-(5,6,7-trihydro-11-methoxybenzo[3,4] cyclohept[2,1-a]indol-13-yl)ethanamine), naltrexone, naltrindole and 5′-guanidinonaltrindole were obtained from Sigma (St. Louis, MO, USA). Streptozotocin was freshly dissolved in distilled water (obtained from a Milli-Q water system), protected from light and immediately administered. Melatonin was dissolved in carboxymethyl cellulose 1% and given orally by a gastric tube at a volume ratio of 4 ml/kg. K-185 was dissolved in 20% dimethylsulfoxide (DMSO) while that naltrexone, naltrindole and 5′-guanidinonaltrindole were dissolved in 0.9% isotonic saline.
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2.6. Study design Independent groups of animals (n = 6) were used for each experimental condition. In order to define the best time to administer melatonin, diabetic rats received an oral administration of vehicle (carboxymethyl cellulose 1%) or melatonin (150 mg/ kg, p.o.) at 20 and 60 min before formalin injection (50 μl). These times were selected from the literature (El-Shenawy et al., 2002) and from pilot experiments in our laboratory. Since the best antinociceptive effect was observed at the 60-min pretreatment (data not shown), dose–response curve for melatonin was carried out giving vehicle or increasing doses of melatonin (10–300 mg/ kg, p.o.) 60 min before formalin injection into the right paw. In an attempt to determine the possible mechanism of antinociceptive action of melatonin in diabetic rats, the selective melatonin MT2 receptor antagonist K-185 (0.2–2 mg/kg, s.c.) was administered 15 min before melatonin (150 mg/kg, p.o.) administration. To establish whether melatonin-induced antinociception was mediated by opioid receptors activation, the effect of the non-selective opioid receptor antagonist naltrexone (– 70 min, 1 mg/kg, s.c.) on the antinociceptive activity induced by melatonin (− 60 min, 150 mg/kg, p.o.) was assessed. Furthermore, to define the nature of the opioid receptor involved in the antinociceptive effect of melatonin in diabetic rats, the selective δ (naltrindole, 0.5 mg/kg, s.c.) and κ (5′guanidinonaltrindole, 1 mg/kg, s.c.) opioid receptor antagonists were given 10 min before melatonin (150 mg/kg, p.o.). Melatonin MT2 receptor antagonist K-185 (Faust et al., 2000) and the δ and κ opioid receptor antagonists naltrindole (Portoghese et al., 1988) and 5′-guanidinonaltrindole (Jones and Portoghese, 2000), respectively, were chosen by their high affinity for the studied receptors. In addition, doses of these drugs were selected from previous studies (Reeta et al., 2006; Qi et al., 2006) and from pilot experiments in our laboratory. For the study of allodynia, rats received the oral administration of vehicle (carboxymethyl cellulose 1%) or increasing doses of melatonin (75–300 mg/kg, p.o.) and withdrawal threshold in diabetic rats (4–8 weeks) was measured for the next 4 h. Observer was unaware of the treatment in each animal.
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according to trapezoidal rule. For allodynia, curves were constructed plotting the 50% threshold for paw withdrawal as a function of time. Effect was also expressed as the area under the 50% threshold withdrawal against time curve (AUC). One-way analysis of variance (ANOVA) followed by Tukey's test was used to compare differences between treatments. The Mann–Whitney test was used to assess differences in rats submitted to the rotarod test. Differences were considered to reach statistical significance when P b 0.05. 3. Results 3.1. Streptozotocin injection and formalin-evoked flinching behavior in non-diabetic and diabetic rats Streptozotocin, but not distilled water, injection caused hyperglycemia. The blood glucose level measured in these rats was 61.8 ± 3.7 and 65± 1.9 mg/dl before distilled water, or streptozotocin injection and 89.2 ±0.86 and 368.6± 14.8 mg/dl 15 days after saline or streptozotocin injection (5 rats), respectively. Moreover, these rats had an increase in food and water intake and displayed polyuria (data not shown). Both the streptozotocin-induced diabetic (2 weeks) and nondiabetic groups exposed to 0.5% formalin exhibited the biphasic pattern of this test characterized by periods of flinching of the injected hind paw separated by a period of decreased activity. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats (Fig. 1A). The overall analysis of formalin-evoked flinching as AUC, showed a small but significant difference (P b 0.05) between the diabetic and non-
2.7. Motor co-ordination test Two independent groups of rats (n = 6, each) were examined for motor co-ordination in a treadmill apparatus (rotarod test) before and after receiving melatonin 300 mg/kg or vehicle by oral administration. Animals were placed upon a cylinder (7 cm diameter) rotating at a speed of 10 rpm. Rats were trained to walk on the cylinder for three consecutive sessions and on the fourth, they received the drug or vehicle treatment (time 0) and the number of falls during a 5-min period was counted after 45 min. 2.8. Data analysis and statistics All results are presented as the mean ± S.E.M. for 6 animals per group. For the formalin test, curves were made for the mean number of flinches against time. The area under the number of flinches against time curves (AUC) for both phases was calculated
Fig. 1. A) Time course of the nociceptive behavior induced by subcutaneous injection of 0.5% formalin to non-diabetic (white circles) and diabetic (black circles) rats. Panels B and C show formalin-induced nociceptive behavior, in non-diabetic and diabetic rats during phases 1 and 2, respectively. The later data are expressed as the area under the number of flinches against time curve (AUC). Data are the mean ± S.E.M. of 6 animals. ⁎Significantly different from the nondiabetic group (P b 0.05), as determined by Student′s t-test.
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diabetic groups during the first phase (Fig. 1B). Moreover, additional analysis revealed that the diabetic group had significantly (P b 0.05) increased frequency of flinching during the second phase (Fig. 1C). Since streptozotocin mainly increased nociception during phase 2 of the formalin test, in the following studies only phase 2 was further analyzed. 3.2. Antinociceptive effect of melatonin in diabetic rats Oral administration of melatonin (150 mg/kg) significantly (P b 0.05) reduced formalin-induced nociceptive behavior at 20and 60-min pretreatment. However, the best antinociceptive effect was observed with the 60-min pretreatment (data not shown) and this pretreatment time was used in the following experiments. Under this condition, melatonin significantly (Fig. 2A, P b 0.05) and dose-dependently (Fig. 2B) reduced flinching behavior during phase 2 in diabetic rats. 3.3. Effect of K-185 and naltrexone on the antinociceptive effect of melatonin in diabetic rats Subcutaneous administration of K-185 (0.2–2 mg/kg) blocked in a dose-related manner the melatonin-induced antinociceptive activity in diabetic rats (Fig. 3A). Moreover, naltrexone (1 mg/kg, s.c.) partially reduced the antinociceptive effect of melatonin in diabetic rats (2 weeks, Fig. 3B). At the greatest tested doses, K-185 or naltrexone did not modify the formalin-induced nociception in diabetic rats (Fig. 3).
Fig. 3. Effect of the melatonin MT2 receptor antagonist K-185 (A) and the nonselective opioid receptor antagonist naltrexone (NTX, B) on the antinociceptive effect of melatonin in diabetic rats submitted to the 0.5% formalin test. Data are expressed as the area under the curve of the number of flinches against time (AUC) of phase 2. Bars are the mean ± S.E.M. for 6 animals. ⁎Significantly different from the vehicle group (P b 0.05) and #significantly different from melatonin group, as determined by analysis of variance followed by Tukey's test.
3.4. Effect of naltrindole and 5′-guanidinonaltrindole on the antinociceptive effect of melatonin in diabetic rats Subcutaneous administration of the selective δ (naltrindole) and κ (5′-guanidinonaltrindole) opioid receptor antagonists did not modify formalin-induced flinching behavior in diabetic rats (Fig. 4). However, naltrindole partially but significantly (P b 0.05) diminished the antinociceptive effect of melatonin in diabetic rats (Fig. 4A). In marked contrast, the selective κ opioid receptor antagonist 5′-guanidinonaltrindole was not able to modify the antinociceptive effect of melatonin in diabetic rats (Fig. 4B). 3.5. Antiallodynic effect of melatonin in diabetic rats
Fig. 2. A) Time course of the antinociceptive effect of melatonin (300 mg/kg, p. o.) in diabetic rats submitted to the subcutaneous injection of 0.5% formalin. Panel B shows the dose–response curve for the antinociceptive effect of melatonin in diabetic rats. In this plot, data are expressed as the area under the curve of the number of flinches against time (AUC) of phase 2. Bars are the mean ± S.E.M. for 6 animals. ⁎Significantly different from the vehicle group (P b 0.05), as determined by analysis of variance followed by Tukey's test.
Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats compared to the distilled water-injected rats. No autotomy behavior was ever observed during the experiment. On this condition, oral administration of melatonin (300 mg/kg), but not vehicle (carboxymethyl cellulose 1%), increased the withdrawal threshold in diabetic rats (Fig. 5A). The maximal antiallodynic effect was reached at 1.5 h and it lasted for 4 h with the greatest dose tested (300 mg/kg). In addition, melatonin dose-dependently reduced streptozotocin-induced tactile allodynia (P b 0.05) (Fig. 5B).
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Moreover, melatonin is able to reduce tactile allodynia induced by ligation of L5/L6 spinal nerves (Ambriz-Tututi and GranadosSoto, in press) and by thermal hyperalgesia in neuropathic mice with partial tight ligation of the sciatic nerve (Ulugol et al., 2006). Thus, our data confirm these studies and extend these observations by showing that melatonin is able to reverse formalininduced flinching behavior, tactile allodynia and abnormal pain processing mechanisms present in diabetic rats. To our knowledge, this is the first report about the antinociceptive and antiallodynic effect of melatonin in streptozotocin-injected diabetic rats. 4.2. Effect of K-185, naltrexone, naltrindole or 5′guanidinonaltrindole on the antinociceptive effect of melatonin in diabetic rats
Fig. 4. Effect of the selective δ (A) and κ (B) opioid receptor antagonists naltrindole and 5′-guanidinonaltrindole (GNTI), respectively, on the antinociceptive effect of melatonin in diabetic rats submitted to the 0.5% formalin test. Data are expressed as the area under the curve of the number of flinches against time (AUC) of phase 2. Bars are the mean ± S.E.M. for 6 animals. ⁎Significantly different from the vehicle group (P b 0.05) and #significantly different from melatonin group, as determined by analysis of variance followed by Tukey's test.
The mechanisms of antinociceptive and antiallodynic activity of melatonin in diabetic rats are unknown. In an attempt to find the possible mechanisms of antinociceptive action, the effect of K-185 and naltrexone on the antinociceptive effect of melatonin in diabetic rats submitted to the 0.5% formalin test was assessed. The antinociceptive effect of melatonin was diminished by K-185. Since K-185 is a highly selective melatonin MT2 receptor antagonist (Faust et al., 2000),
3.6. Effect of melatonin on motor co-ordination test Animals did not have a fall from the rotarod test before administration of melatonin or saline. Oral administration of melatonin (300 mg/kg) produced 1 fall in 1 of 6 rats during the rotarod test. Moreover, oral administration of vehicle showed the same pattern (data not shown). 4. Discussion 4.1. Antinociceptive and antiallodynic activity of melatonin in diabetic rats In this study we have shown that oral administration of melatonin is able to reduce formalin-induced nociceptive behavior and tactile allodynia in diabetic rats. This antinociceptive and antiallodynic effect was independent of any motor effect as administration of the greatest systemic tested dose (300 mg/kg, p.o.) significantly reduced formalin-induced nociception and tactile allodynia without affecting co-ordination in the rotarod test, as previously reported (Tu et al., 2004; Ambriz-Tututi and Granados-Soto, in press). Several studies have shown that melatonin produces antinociception in inflammatory pain models (Cuzzocrea et al., 1997; Raghavendra et al., 2000; Bilici et al., 2002; El-Shenawy et al., 2002; Ray et al., 2004; Tu et al., 2004).
Fig. 5. A) Time course of the antiallodynic effect of melatonin (300 mg/kg, p.o.) in streptozotocin-pretreated diabetic rats. After diabetes induction, animals were allowed to develop tactile allodynia for 4 to 8 weeks. Animals were treated with oral melatonin and withdrawal threshold was measured for the next 4 h. B) Antiallodynic effect produced by oral administration of melatonin in diabetic (4–8 weeks) rats. Data are expressed as the 50% threshold withdrawal against time curve (AUC). ⁎Significantly different from the vehicle (Veh) group (P b 0.05), as determined by analysis of variance followed by the Tukey's test. In both plots data are the mean ± S.E.M. for 6 animals.
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our data suggest that melatonin-induced antinociception could be due to activation of melatonin MT2 receptors. These results agree with the presence of melatonin MT2 receptors in the dorsal horn of the lumbar spinal cord (Zahn et al., 2003). In addition, our data agree with an inhibitory role for melatonin MT2 receptors as there is evidence that binding of melatonin to melatonin MT2 receptors decreases intracellular concentrations of cyclic AMP, diacylglycerol and arachidonic acid (Iuvone and Gan, 1994; Vanecek, 1998). On the other hand, subcutaneous administration of the nonselective opioid receptor antagonist naltrexone partially diminished the antinociceptive effect induced by the oral administration of melatonin. These data agree with previous observations showing that melatonin-induced antinociceptive and antihyperalgesic (Mantovani et al., 2006) or antiallodynic (Ambriz-Tututi and Granados-Soto, in press) effects in nondiabetic rats can be blocked by the non-selective opioid receptor antagonist naloxone or naltrexone, respectively. In an attempt to define the nature of the opioid receptors activated after oral administration of melatonin, the selective δ and κ opioid receptor antagonists naltrindole (Portoghese et al., 1988) and 5′guanidinonaltrindole (Jones and Portoghese, 2000), respectively, were used alone or in combination with melatonin in diabetic rats. Likewise naltrexone, naltrindole, but not 5′-guanidinonaltrindole, partially reduced melatonin-induced antinociception. Accordingly, it has been demonstrated that intraperitoneal or intracerebroventricular administration of melatonin significantly enhances the δ opioid receptor agonist deltorphin I-induced antinociception, but not that induced by the μ opioid receptor agonist endomorphin-1 (Li et al., 2005). This effect was blocked by the melatonin MT2 receptor antagonist luzindol suggesting that melatonin can augment antinociception induced by the δ opioid receptor agonists acting on melatonin MT2 receptors. Taken together data suggest the possible participation of δ, but not κ, opioid receptors in the antinociceptive effect of melatonin in diabetic rats. It is well known that melatonin is not able to bind opioid receptors (Shavali et al., 2005). However, several observations, including our data, point to significant interactions between melatonin and opioid peptides (Shavali et al., 2005). Melatonin releases β-endorphin, an endogenous opioid peptide, from mouse pituitary cells in culture (Shavali et al., 2005). Hence, it has been suggested that melatonin exerts its antinociceptive action in diabetic rats not by binding to opioid receptors but by inducing β-endorphin release. β-endorphin could then bind to δ (Aloyo and Pazdalski, 1995) or μ (Gilmore and Weiner, 1989) opioid receptors in spinal cord and supraspinally (periaqueductal gray matter) in order to produce its antinociceptive effect. This suggestion agrees with evidence showing that β-endorphin administered i.c.v. produces a naltrindolesensitive antinociceptive effect in diabetic mice (Kamei et al., 1993). In addition, the antinociceptive effect of melatonin in spinal and supraspinal sites in non-diabetic rats also supports this suggestion (Yu et al., 2000a,b; Tu et al., 2004; AmbrizTututi and Granados-Soto, in press). All these sites would be reached considering the high lipid-solubility of melatonin. The final effect would be the opening of K+ channels and closing of
Ca2+ channels (Moises et al., 1994; Kieffer, 1995; Lohmann and Welch, 1999) leading to cell hyperpolarization and reduction of pain. In this study we have provided evidence about the possible mechanisms involved in the antinociceptive actions of melatonin in diabetic rats submitted to the 0.5% formalin test. It is likely that some of these mechanisms could work to explain the antiallodynic activity of this drug. However, the experiments demonstrating these mechanisms are still lacking. 4.3. Final considerations Diabetes-induced hyperglycemia plays a critical role in the development and progression of diabetic neuropathy. One of the mechanisms by which hyperglycemia causes neural degeneration is via the increased oxidative stress that accompanies diabetes (Montilla et al., 1998; Baydas et al., 2003; Nishida, 2005). Hyperglycemia causes a reduction in the levels of protective endogenous antioxidants and increases generation of free radicals (Baydas et al., 2002). Contrariwise, melatonin is a direct scavenger of free radicals and has indirect antioxidative effects due to the stimulation of the expression and activity of antioxidative enzymes such as glutathione peroxidase, superoxide dismutase and catalase in mammalian cells (Nishida, 2005; Klepac et al., 2006). Therefore, it is tempting to suggest that these actions could explain at least part of the antinociceptive and/or antiallodynic effect in diabetic rats. The fact that systemic administration of free radical scavengers is able to reduce tactile allodynia in the spinal nerve ligation model in rats (Kim et al., 2004) agrees with our suggestion. In conclusion, oral administration of melatonin to diabetic rats significantly reduces formalin-induced nociception and tactile allodynia. The antinociceptive effects are completely prevented by the melatonin MT2 receptor antagonist K-185 and partially prevented by the non-selective opioid receptor antagonist naltrexone and the selective δ opioid receptor antagonist naltrindole, but not by the selective κ opioid receptor antagonist 5′-guanidinonaltrindole. Thus, data suggest the possible participation of melatonin MT2 and δ opioid receptors in the antinociceptive activity of melatonin in diabetic rats. Some of these mechanisms, along with the ability to scavenge free radicals, could also explain the antiallodynic effect shown by this drug. Finally, our data suggest that melatonin could be a promising drug to treat diabetic patients. Acknowledgements This work was done at the Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados, Sede Sur, México, D.F., Mexico. Authors greatly appreciate the technical assistance of Guadalupe C. Vidal-Cantú and bibliographic assistance of Héctor Vázquez. Rosaura Arreola-Espino, Mónica Ambriz-Tututi, Claudia I. Araiza-Saldaña, Nadia L. CaramSalas and Héctor I. Rocha-González are CONACYT fellows. In addition, Claudia I. Araiza-Saldaña and Nadia L. Caram-Salas are the recipients of the “Apoyos Integrales para la Formación de Doctores en Ciencias” fellowship. Teresa Mixcoatl-Zecuatl is a
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